The artifactual nature of stavudine binding to human serum albumin. A fluorescence quenching and isothermal titration calorimetry study

The interaction between stavudine, a nucleoside reverse transcriptase inhibitor and human serum albumin (HSA), was investigated by fluorescence quenching technique and isothermal titration calorimetry (ITC). A good linearity of albumin fluorescence quenching in the presence of stavudine was determined. Analyzing these data we obtained for the dissociation constant the value Kd = (18.18 ± 0.46) × 10−5 M. However, due to contradictory results obtained in ITC experiments, we checked the fluorescence quenching data for the inner-filter effect, the main confounding factor in the observed quenching. Based on the UV–vis absorption data we have corrected the observed fluorescence intensities and concluded, in accordance with ITC results, that stavudine binding to HSA is negligible and the observed quenching effect is entirely caused by a failure to correct for the inner-filter effect.

► The interaction between stavudine and HSA was investigated by fluorescence quenching technique and ITC.
► The contradictory results provided by these techniques were clarified by taking into account the inner filter effect.
► We show that the recently reported association constant between stavudine and HSA is due to an experimental artifact.